A determination method of the type mentioned above is based on the reversible binding of cations to a cation-selective ionophore and the so-called "PET effect" between the ionophore and a luminophoric moiety.
The so-called "PET effect" denotes the transfer, induced by photons, of electrons (photoinduced electron transfer=PET) from the ionophoric moiety or ionophore, respectively, to the luminophoric moiety or luminophore, respectively, which leads to a decrease in the (relative) luminescence intensity and the luminescence decay time of the luminophore. Absorption and emission wavelengths, however, remain basically unaffected in the process (J. R. Lakowicz in "Topics in Fluorescence Spectroscopy", Volume 4: Probe Design and Chemical Sensing; Plenum Press, New York & London (1994)).
By the binding of ions to the ionophore, the PET effect is partially or completely inhibited, so that there is an increase in the relative luminescence intensity and an increase in the luminescence decay time of the luminophoric moiety. Hence, the concentration or the activity of the ion to be determined can be deduced by measuring the luminescence properties, i.e., relative luminescence intensity and/or luminescence decay time. Activities can be related to concentrations via known Debye-Huckel formalisms.
It is known that cryptands preferably form complexes (cryptates) with such cations whose ion radius corresponds as well as possible to that of the cavity formed by the cryptand (Lehn J M, Sauvage J P, Amer. Chem. Soc. 97, 6700-6207, 1975). The ion diameters of the alkali metals Li, Na, K and Rb are 0.78, 0.98, 1.33 and 1.49 Angstrom, respectively. Thus, for a given cryptand the selectivity for a particular cation can be adjusted by changes in the ether chains. Furthermore, it is known that cryptands having indicator properties can be obtained by the coupling of cryptands to chromophores or luminophores.
A method of the kind initially described is known from U.S. Pat. No. 5,439,828, wherein diaza-cryptands are utilized as the luminophore-ionophore, which diaza-cryptands have been functionalized as fluorophores with fluorescent coumarins and, depending on their structure, are selective for lithium, sodium and potassium ions, respectively. It is stated that these luminophore-ionophores can be used in sample media of neutral pH and are even the preferred choice in such systems.
Yet, research (Frank Kastenholz, Inaugural Dissertation, University of Cologne, 1993, FIG. 32, p. 54) has shown that in the physiological pH range the fluorescence signal depends significantly on the pH of the sample and increases considerably with a decreasing pH, even from pH 7.4 onwards. This affects the accuracy of a determination carried out in a biological sample. Moreover, the compounds used have the additional disadvantage that the employed coumarins show absorption wavelengths of about 336 nm and hence cannot be excited by commercial LEDs.
These disadvantages also apply to the luminophore-ionophores mentioned in U.S. Pat. No. 5,162,525.
From DeSilva, Tetrahedron Letters, Volume 31, No. 36, pp. 5193-5196 (1990), diaza-cryptands are known in which the two nitrogen atoms are each bound to a respective aromatic ring, i.e., both bridging nitrogens are aryl nitrogens. Research conducted by the applicant has shown that these diaza-cryptands are not suited for determining potassium ions via a PET mechanims. By the binding of K.sup.+ to these diaza-cryptands in the abscence or in the presence of physiological Na.sup.+ concentrations, the enhancement of the luminescence intensity of the fluorophore moiety (i.e., napthalimide) due to the PET effect is too small for a useful practical method.
From EP-A-0,881,488 diaza-cryptands are known in which one of the two bridging nitrogen atoms is an aryl nitrogen and the other one is an aliphatic nitrogen. From the perspective of synthesis, the production of those cryptands in quantities required for commercial use is expensive. EP-A-0,881,488 suggests the Williamson ether synthesis to make the precursors of the crown ether. The oily precursors are tedious to purify and the cyclization reactions give low yields. The overall yield of the synthetic path is low.
The present invention, therefore, has as its object to improve the known process and make available luminophore-ionophores which lack significant dependence of the luminescence properties on the pH value of the sample at physiological pH values and, thus, preferably are suitable for determining alkali ions in biological samples. In addition, the invention aims at providing luminophore-ionophores which are particularly well suited for use in the determination of K.sup.+ ions in a sample. Further, the method of the invention is to be particularly well suited for practice in the determination of an alkali ion in the presence of physiological concentrations of other alkali ions, i.e., it should exhibit a strong dependence of the luminescent signal on the concentration of the alkali ion being determined.